Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43

Abstract

TDP-43 is a multifunctional DNA/RNA-binding protein that has been identified as the major component of the cytoplasmic ubiquitin (+) inclusions (UBIs) in diseased cells of frontotemporal lobar dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). Unfortunately, effective drugs for these neurodegenerative diseases are yet to be developed. We have tested the therapeutic potential of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) and three other autophagy activators (spermidine, carbamazepine, and tamoxifen) in a FTLD-U mouse model with TDP-43 proteinopathies. Rapamycin treatment has been reported to be beneficial in some animal models of neurodegenerative diseases but not others. Furthermore, the effects of rapamycin treatment in FTLD-U have not been investigated. We show that rapamycin treatment effectively rescues the learning/memory impairment of these mice at 3 mo of age, and it significantly slows down the age-dependent loss of their motor function. These behavioral improvements upon rapamycin treatment are accompanied by a decreased level of caspase-3 and a reduction of neuron loss in the forebrain of FTLD-U mice. Furthermore, the number of cells with cytosolic TDP-43 (+) inclusions and the amounts of full-length TDP-43 as well as its cleavage products (35 kDa and 25 kDa) in the urea-soluble fraction of the cellular extract are significantly decreased upon rapamycin treatment. These changes in TDP-43 metabolism are accompanied by rapamycin-induced decreases in mTOR-regulated phospho-p70 S6 kinase (P-p70) and the p62 protein, as well as increases in the autophagic marker LC3. Finally, rapamycin as well as spermidine, carbamazepine, and tamoxifen could also rescue the motor dysfunction of 7-mo-old FTLD-U mice. These data suggest that autophagy activation is a potentially useful route for the therapy of neurodegenerative diseases with TDP-43 proteinopathies.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Phenotype characterizations of rapamycin-treated FTLD-U mice. (A) Flowchart of rapamycin treatment of mice. WT and TDP-43 Tg mice were treated with vehicle or rapamycin as described in Materials and Methods. Treatment continued from the age of 2 mo to the age of 6 mo. Water maze tests (I and II) were carried out at the ages of 2 mo and 3 mo, respectively, and the rotarod test was carried out at the age of 6 mo. (B) Water maze performances of 3-mo-old WT and TDP-43 Tg mice after treatment with rapamycin or vehicle for 1 mo. Note the rescued performance of the Tg mice in the hidden platform tasks (Left) after treatment with rapamycin (Tg-Rapa) in comparison with the ones treated with vehicle (Tg-Veh). Visible platform tasks (Right) of the mice were examined after the hidden platform tasks. Mixed-design ANOVA on the escape latencies across the day indicates the significance of comparisons between the sessions (within-subjects factor, F(3.36, 53.74) = 442.62; P < 0.0001) and the groups (between-subjects factor, (F(3,16) = 249.4; P < 0.0001). Results in B represent the mean ± SEM of three independent experiments (n = 10 male mice per group). (C) Rotarod performances of 6-mo-old WT and TDP-43 Tg mice after treatment with rapamycin or vehicle for 4 mo. Note the better performance of the Tg-Rapa mice than the Tg-Veh mice. Results in C represent the mean ± SEM of three independent experiments (n = 10 male mice per group). *P < 0.05.

Fig. 2.

Rapamycin effects on caspase-3 expression and neuronal survival in FTLD-U mouse brains. (A) Western blotting analysis of the levels of pro- and active forms of caspase-3 in the extracts of isolated cortex and hippocampus from 6-mo-old WT and TDP-43 Tg mice treated with rapamycin or vehicle. Blotting patterns are shown on the Left and the statistical analysis is shown on the Right. Results are representative of the mean ± SEM of three independent experiments (n = 5 mice per group). *P < 0.05. (B) Measurement of the caspase-3 activities in the extracts from the cortex and hippocampus of TDP-43 Tg mice treated with rapamycin (Rapa)- or vehicle (Veh) by fluorimetric assay. Histogram represents caspase-3 activities (mean ± SEM) of three independent experiments (n = 3 mice per group). *P < 0.05. (C) Immunohistostaining analysis of active caspase-3 (Top two panels) in the cortex of 6-mo-old TDP-43 Tg mouse brains treated with rapamycin (Rapa) or vehicle (Veh) for 4 mo. Quantitative analysis by tissue cytometry using TissueQuest software is shown in the Middle and the Bottom histograms. (D) Immunohistostaining of NeuN(+) cells in the cortex of 6-mo-old TDP-43 Tg mice treated with rapamycin (Rapa) or vehicle (Veh) for 4 mo (Top two panels). Quantitative analysis is shown in the Middle two panels and the Bottom histogram. Results in both C and D represent the mean ± SEM of three independent experiments (n = 5 mice per group). (Bars, 25 μm.) *P < 0.05.

Fig. 3.

Rapamycin effects on the solubility and subcellular distribution of TDP-43 in FTLD-U mouse brains. (A) Western blot analysis of TDP-43 in the soluble/ RIPA fractions of extracts from the cortex and hippocampus regions of 6-mo-old WT (Upper set of panels and histogram) and TDP-43 Tg mice (Lower set of panels and histogram) treated with rapamycin or vehicle. The RIPA fractions of the extracts were prepared as described in Materials and Methods and analyzed by Western blotting. Note that rapamycin and vehicle treatments resulted in similar levels of the different TDP-43 species in either the WT or the Tg mouse samples. Amounts of the 35-kDa and 25-kDa TDP-43 fragments in the WT extract were too low to be analyzed. (B) Western blot analysis of TDP-43 in the urea-soluble fractions of brain extracts. The arrow points to the unmodified form of TDP-43 on the gel. The triangle is an anti–TDP-43 hybridizing band of unknown origin. ***, represents the gel region containing high molecular weight, polyubiquitinated TDP-43 species. Result is representative of three independent experiments. (C) Immunohistochemical staining of TDP-43 and nuclei of brain sections from TDP-43 Tg mice treated with rapamycin or vehicle. Sections were immunohistochemically stained for detection of TDP-43 (brown) and nuclei (blue) as described in Materials and Methods. TDP-43 (+) inclusions are indicated by white arrows. One cell each from the two images are further magnified in the Lower Right corners for better visualization. (Bars, 10 μm.) Quantitative analysis of the relative numbers of TDP-43 (+) inclusions is shown in the histogram below the photo panels. Results in A and C represent the mean ± SEM of three independent experiments (n = 5 mice per group). *P < 0.05.

Fig. 4.

Effects of rapamycin treatment on autophagy in the forebrains of WT and TDP-43 Tg mice. (A, Left) representative immunofluorescence images of the hippocampus of TDP-43 Tg mice treated with rapamycin/vehicle and costained with anti-LC3 (green)/DAPI (blue). Note the increase of the LC3 puncta in the rapamycin-treated Tg mouse sample in comparison with the vehicle-treated one. (Bars, 10 μm.) (Right) Histogram of quantification of LC3 immunofluorescence. Result is representative of three independent experiments (n = 5 sections per group). *P < 0.05. (B) Western blot analysis of expression levels of LC3, p62, P-p70, and p70 S6 kinase. Levels of proteins and tubulin control in the extracts of the isolated cortex and hippocampus from WT and TDP-43 Tg mice with or without rapamycin treatment were measured by Western blotting. (a) Blot patterns. Comparisons among WT and Tg mice treated with rapamycin (Rapa) or vehicle (Veh) are shown in the histograms for LC3-II normalized with LC3-I (b), p62 normalized with tubulin (c), and P-p70 normalized with p70 (d), respectively. For all three histograms, the protein levels are first normalized and then compared with WT (Veh) samples. Results represent the mean ± SEM of three independent experiments (n = 5 mice per group). *P < 0.05.

Fig. 5.

Effects of autophagy activation in the rescue of motor dysfunction of 7-mo-old CamKII–TDP-43 Tg mice. (A) Rotarod tests of 7-mo-old CamKII–TDP-43 Tg mice after treatment with rapamycin, spermidine, carbamazepine, tamoxifen, or vehicle for 1 mo, as described in Materials and Methods. As the control, FVB/NJNarl-WT mice treated with rapamycin or vehicle at the same age were also tested. Note the improvement of the rotarod performance of the TDP-43 Tg mice upon treatment with each of the four chemicals. Data represent the mean ± SEM of three independent experiments (n = 10 mice per group). *P < 0.05. (B) Quantitative comparison of the relative abundance of NeuN(+) cells in the brain sections of 7-mo-old TDP-43 Tg mice treated with rapamycin, spermidine, carbamazepine, tamoxifen, or vehicle for 1 mo. The number of NeuN(+) cells of the vehicle samples is taken as 100 (n = 5 mice per group). *P < 0.05. (C) Histograms showing quantitative analysis of the relative numbers of TDP-43 (+) inclusion-containing cells in the cortex sections from 7=mo-old TDP-43 Tg mice that were treated with rapamycin, spermidine, carbamazepine, tamoxifen, or vehicle for 1 mo. The number of vehicle samples is taken as 100. Results in B and C represent the mean ± SEM of three independent experiments (n = 5 mice per group). *P < 0.05. (D) Western blot analysis of expression levels of LC3-I, LC3-II, and p62. Levels of proteins in the extracts of isolated cortex and hippocampus from 7-mo-old WT and TDP-43 Tg mice with or without drug treatment were analyzed and compared. (a) Representative Western blot. (b) Histogram comparing the ratios of LC3-II/LC3-I in different samples. (c) Histogram comparing the ratios of p62/tubulin in different samples. In both b and c, the ratios of WT (Veh) samples were taken as 100. The altered level of LC3-II and the accumulation of p62 in Tg mice relative to WT mice indicate the impairment of autophagy, which has also been observed in a SOD-1 (G93A) Tg mouse model (28). Results represent the mean ± SEM of three independent experiments (n = 5 mice per group). *P < 0.05.